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Kinetochore-independent chromosome poleward movement during anaphase of meiosis II in mouse eggs.

Deng M, Gao J, Suraneni P, Li R - PLoS ONE (2009)

Bottom Line: Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores.Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads.The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America. MQD@stowers-institute.org

ABSTRACT
Kinetochores are considered to be the key structures that physically connect spindle microtubules to the chromosomes and play an important role in chromosome segregation during mitosis. Due to different mechanisms of spindle assembly between centrosome-containing mitotic cells and acentrosomal meiotic oocytes, it is unclear how a meiotic spindle generates the poleward forces to drive two rounds of meiotic chromosome segregation to achieve genome haploidization. We took advantage of the fact that DNA beads are able to induce bipolar spindle formation without kinetochores and studied the behavior of DNA beads in the induced spindle in mouse eggs during meiosis II. Interestingly, DNA beads underwent poleward movements that were similar in timing and speed to the meiotic chromosomes, although all the beads moved together to the same spindle pole. Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores. Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads. The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

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Role of cytoplasmic dynein in chromosome poleward movement.(A) Inhibition of poleward movement of DNA beads and maternal chromosomes by dynein antibody injection. (B) Injection of dynamitin p50. Note that DNA beads remained at the middle of the spindles (arrows) whereas the maternal chromosomes segregated and moved to spindle poles (arrowheads). (C) Quantification of the percentage of chromosome poleward movements on the meiotic and DNA bead spindles respectively in the dynein antibody-injected eggs. (D) Dynein localization on DNA beads (arrow). (E) Merged image of DNA beads (blue) and spindle (green). (F) Dynein localization on MII chromosomes (arrow) and spindle poles (arrowheads). (G) Merged image of DIC and DAPI staining showing MII chromosomes (blue) and spindle. (H–K) p-MARCKS staining of microtubule organization centers (MTOCs, red) in DNA bead spindle (H and I) and MII spindle (J and K). Note that there is only one MTOC staining in each of the DNA bead-spindle poles (H, arrows). (L–O) p150Glued localization on both DNA beads (H and I, arrows) and meiotic chromosomes (J and K, arrows).
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pone-0005249-g004: Role of cytoplasmic dynein in chromosome poleward movement.(A) Inhibition of poleward movement of DNA beads and maternal chromosomes by dynein antibody injection. (B) Injection of dynamitin p50. Note that DNA beads remained at the middle of the spindles (arrows) whereas the maternal chromosomes segregated and moved to spindle poles (arrowheads). (C) Quantification of the percentage of chromosome poleward movements on the meiotic and DNA bead spindles respectively in the dynein antibody-injected eggs. (D) Dynein localization on DNA beads (arrow). (E) Merged image of DNA beads (blue) and spindle (green). (F) Dynein localization on MII chromosomes (arrow) and spindle poles (arrowheads). (G) Merged image of DIC and DAPI staining showing MII chromosomes (blue) and spindle. (H–K) p-MARCKS staining of microtubule organization centers (MTOCs, red) in DNA bead spindle (H and I) and MII spindle (J and K). Note that there is only one MTOC staining in each of the DNA bead-spindle poles (H, arrows). (L–O) p150Glued localization on both DNA beads (H and I, arrows) and meiotic chromosomes (J and K, arrows).

Mentions: To gain insights into the forces responsible for driving the kinetochore-independent poleward movements of DNA beads during anaphase, we tested the potential role of two microtubule motors using chemical or protein inhibitors. Whereas we were unable to obtain conclusive results with inhibition of Eg5 kinesin by monastrol due to severe defects in maintaining a bipolar spindle morphology in anaphase (data not shown), we found that dynein, a minus end-directed microtubule motor, was required for the anaphase poleward movement of DNA beads, as injection of dynein functional-blocking antibody, clone 70.1, completely blocked the poleward movement of DNA beads (Figure 4A, arrow, n = 17, movie S2). To verify if the inhibition by anibody injection is due to specific disruption of dynein function, we performed separate experiment to inject dynamitin p50, a dynein inhibitory peptide [36] and observed the same inhibition of DNA bead poleward movement (Figure 4B, n = 23). The block was not due to possible activation of spindle assembly checkpoint which may arrest cell cycle progression because the maternal meiotic spindle in the same eggs underwent normal metaphase-anaphase transition and chromosome segregation (Figure 4A–4C, arrowheads). It is interesting to note that disruption of dynein function had no effect on the bipolar segregation of the meiotic chromosomes (Figure 4A and 4B, arrowheads) which contain kinetochores. The differential effects of inhibition of dynein on the behavior of kinetochore-free DNA beads and kinetochore-containing meiotic chromosomes after anaphase onset are summarized in Figure 4C.


Kinetochore-independent chromosome poleward movement during anaphase of meiosis II in mouse eggs.

Deng M, Gao J, Suraneni P, Li R - PLoS ONE (2009)

Role of cytoplasmic dynein in chromosome poleward movement.(A) Inhibition of poleward movement of DNA beads and maternal chromosomes by dynein antibody injection. (B) Injection of dynamitin p50. Note that DNA beads remained at the middle of the spindles (arrows) whereas the maternal chromosomes segregated and moved to spindle poles (arrowheads). (C) Quantification of the percentage of chromosome poleward movements on the meiotic and DNA bead spindles respectively in the dynein antibody-injected eggs. (D) Dynein localization on DNA beads (arrow). (E) Merged image of DNA beads (blue) and spindle (green). (F) Dynein localization on MII chromosomes (arrow) and spindle poles (arrowheads). (G) Merged image of DIC and DAPI staining showing MII chromosomes (blue) and spindle. (H–K) p-MARCKS staining of microtubule organization centers (MTOCs, red) in DNA bead spindle (H and I) and MII spindle (J and K). Note that there is only one MTOC staining in each of the DNA bead-spindle poles (H, arrows). (L–O) p150Glued localization on both DNA beads (H and I, arrows) and meiotic chromosomes (J and K, arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664963&req=5

pone-0005249-g004: Role of cytoplasmic dynein in chromosome poleward movement.(A) Inhibition of poleward movement of DNA beads and maternal chromosomes by dynein antibody injection. (B) Injection of dynamitin p50. Note that DNA beads remained at the middle of the spindles (arrows) whereas the maternal chromosomes segregated and moved to spindle poles (arrowheads). (C) Quantification of the percentage of chromosome poleward movements on the meiotic and DNA bead spindles respectively in the dynein antibody-injected eggs. (D) Dynein localization on DNA beads (arrow). (E) Merged image of DNA beads (blue) and spindle (green). (F) Dynein localization on MII chromosomes (arrow) and spindle poles (arrowheads). (G) Merged image of DIC and DAPI staining showing MII chromosomes (blue) and spindle. (H–K) p-MARCKS staining of microtubule organization centers (MTOCs, red) in DNA bead spindle (H and I) and MII spindle (J and K). Note that there is only one MTOC staining in each of the DNA bead-spindle poles (H, arrows). (L–O) p150Glued localization on both DNA beads (H and I, arrows) and meiotic chromosomes (J and K, arrows).
Mentions: To gain insights into the forces responsible for driving the kinetochore-independent poleward movements of DNA beads during anaphase, we tested the potential role of two microtubule motors using chemical or protein inhibitors. Whereas we were unable to obtain conclusive results with inhibition of Eg5 kinesin by monastrol due to severe defects in maintaining a bipolar spindle morphology in anaphase (data not shown), we found that dynein, a minus end-directed microtubule motor, was required for the anaphase poleward movement of DNA beads, as injection of dynein functional-blocking antibody, clone 70.1, completely blocked the poleward movement of DNA beads (Figure 4A, arrow, n = 17, movie S2). To verify if the inhibition by anibody injection is due to specific disruption of dynein function, we performed separate experiment to inject dynamitin p50, a dynein inhibitory peptide [36] and observed the same inhibition of DNA bead poleward movement (Figure 4B, n = 23). The block was not due to possible activation of spindle assembly checkpoint which may arrest cell cycle progression because the maternal meiotic spindle in the same eggs underwent normal metaphase-anaphase transition and chromosome segregation (Figure 4A–4C, arrowheads). It is interesting to note that disruption of dynein function had no effect on the bipolar segregation of the meiotic chromosomes (Figure 4A and 4B, arrowheads) which contain kinetochores. The differential effects of inhibition of dynein on the behavior of kinetochore-free DNA beads and kinetochore-containing meiotic chromosomes after anaphase onset are summarized in Figure 4C.

Bottom Line: Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores.Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads.The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America. MQD@stowers-institute.org

ABSTRACT
Kinetochores are considered to be the key structures that physically connect spindle microtubules to the chromosomes and play an important role in chromosome segregation during mitosis. Due to different mechanisms of spindle assembly between centrosome-containing mitotic cells and acentrosomal meiotic oocytes, it is unclear how a meiotic spindle generates the poleward forces to drive two rounds of meiotic chromosome segregation to achieve genome haploidization. We took advantage of the fact that DNA beads are able to induce bipolar spindle formation without kinetochores and studied the behavior of DNA beads in the induced spindle in mouse eggs during meiosis II. Interestingly, DNA beads underwent poleward movements that were similar in timing and speed to the meiotic chromosomes, although all the beads moved together to the same spindle pole. Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores. Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads. The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

Show MeSH